The lactose (lac) operon consists of three protein products under the control of a lac promoter-operator. These gene products are .beta.-galactosidase (Z), permease (Y), and thiogalactoside transacetylase (A). The protein product of the lacI transcript (repressor), an independent gene product, interacts with the operator of the lac operon and keeps synthesis off until allolactose (1,6-0-.beta.-D-galactopyranosyl-D-glucose), a product of the .beta.-galatosidase reaction, accumulates in the cell and binds to the repressor. The allolactose repressor complex has a changed conformation, allowing the repressor to be displaced from the operator. RNA transcription then begins from the lac promoter (Beckwith, J. [1987] In Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology. Vol. 2, Neidhardt, F. C., Editor in Chief, American Society for Microbiology, Washington, D.C.).
A few molecules of the lac operon transcript are present in E. coli, even in the absence of lactose. Hence, permease and .beta.-galactosidase are always present, at least at a low level. Allolactose, the natural inducer of the operon, is made in the cell when lactose (1,4-0-.beta.-D-galactopyanosyl-D-glucose) enters the cell by the permease reaction and is converted through transgalactosidation by .beta.-galactosidase into allolactose (Freifelder, D., [1987] Molecular Biology. Jones and Bartlett Publishers, Inc., Portola Valley, CA; Beckwith, supra). It was calculated that greater than 20% of the lactose acted upon by .beta.-galactosidase is converted to allolactose (Jobe, A., Bourgeois, S., [1972] J. Mol. Biol. 69:397-408). The majority of the remaining lactose is converted to glucose and galactose. Allolactose is a better substrate for .beta.-galactosidase than lactose (Jobe and Bourgeois, supra), and is itself rapidly converted to glucose and galactose.
Another control element of the lac operon is catabolite repression. In the presence of glucose the cell is able to repress many operons. For example the lac operon is only transcribed at 2% of its maximum level in the presence of glucose (Beckwith, supra).
When several .beta.-galactosides were compared for their ability to induce the lac operon in vivo, lactose was found to act as a very poor effector molecule. The synthetic non-metabolized .beta.-galactoside, isopropyl-.beta.-D-thiogalactoside (IPTG), was found to induce the lactose operon 5 times better than lactose (Monod, J., G. Cohen-Bazire and M. Cohn [1951] Biochim. Biophys. Acta. 7:585-599). Yet, allolactose itself is as good an inducer of the operon as IPTG (Muller-Hill, B., H. V. Rickenberg and K. Wallenfels [1964] J. Mol. Biol. 10:303-318; Jobe and Bourgeois, supra).
Given the efficiency of lactose conversion to allolactose, one might expect lactose to work very well as an inducer of the lac operon. However, prior to the present disclosure, this has never occurred in practice (Monod et al., supra).
The current way to control the expression of heterologous genes from the lac promoter or lac concensus promoters such as tac (Rezinkoff, W. S. and W. R. McClure [1986] Maximizing Gene Expression, W. Reznikoff and L. Gold, eds., Butterworth Publishers, Stoneham, MA) is to have enough lac repressor present in the cell, so that transcription from the tac promotor is off until IPTG or another proper inducing .beta.-galactoside is added to the cell. Although IPTG is the current inducer of choice, it is expensive and has been labeled a potential carcinogen. Thus, there is a need to replace IPTG in commercial systems where control of the expression of heterologous genes from lac operated promoters is used.